PROJECT SUMMARY Bone pseudoarthrosis is a debilitating defect of bone healing. Both fracture and pseudoarthrosis represent the epiphenomena of an underlying, localized congenital disorder of skeletal tissues that alters the process of bone growth and remodeling and jeopardizes both the development of a mechanically sound internal structure and the process of bone repair. The etiopathology of pseudoarthrosis remains to be defined for the establishment of adapted therapies. Bone healing involves four main cell types: mesenchymal stem cells (MSCs), chondrocytes, osteoblasts and osteoclasts. To date, evidence suggests that lack of Nf1 in bone forming cells is responsible for NF1 (neurofibromatosis) pseudoarthrosis, but the cell types involved remain unidentified. The challenges today are to identify the cell types whose function is impaired in NF1 pseudoarthrosis, to characterize the defects associated with loss of function of Nf1 in this cell type, and based on this knowledge to propose adapted strategies to correct these defects. In this proposal, we ask whether Nf1 haplo-insufficiency or Nf1 loss of function in three specific bone cell types, chondrocytes, osteoblasts or their common precursor, MSCs, impairs bone healing. To address this question, we propose to use Nf1+/- mice and conditional mouse models lacking one or both copies of Nf1 specifically in MSCs, chondrocytes or osteoblasts in bone repair studies. With these mouse models in hand, it will possible to determine whether one or two copies of Nf1 is required for the formation of a cartilaginous callus by chondrocytes or for the calcification and remodeling of this callus by osteoblasts following fracture (Specific Aim I and II). We also designed studies aimed at characterizing a new therapeutic approach for treating bone pseudoarthrosis, based on our previous work (R21AR053978-01) and the ERK inhibitory property of PD19830 and lovastatin to correct the defects of Nf1-/- osteoblasts (Specific Aim III). The studies proposed in this application aim at characterizing novel pathways and genes regulating bone remodeling and repair, with the long-term goals of better understanding skeleton diseases and proposing adapted therapies. We expect our studies 1) to characterize the role of Nf1 at specific stages of bone repair in specific bone cell types, 2) to characterize novel therapeutic targets and strategies to identify the most appropriate treatment for this syndrome and 3) to provide new mouse models that can be used as pre-clinical tools.